Activation of the transcription factor NF-KB by DNA-damaging anticancer agents has emerged as an important modulator of malignant behaviors, including resistance to apoptotic cell death. This signal transduction pathway also serves as an attractive paradigm to understand how nuclear DNA damage induces a signaling pathway to activate a cytoplasmically sequestered transcription factor - a nuclear-to-cytoplasmic signal transduction pathway. The long-term goal of this proposal is to increase our understanding of the mechanisms involved in NF-KB signal transduction pathways initiated by the model DNA-damaging anticancer agents, camptothecin and etoposide. In our published studies, we provided evidence that a convergence of two parallel pathways is induced by these genotoxic agents: (a) DSB-dependent activation of ATM;and (b) a stress signal that leads to nuclear accumulation of NEMO/IKKy, the regulatory subunit of the kB kinase (IKK) complex, via a SUMO-1 modification. Ultimately, NEMO exports in an ATM- and ubiquitin-dependent manner and activates the cytoplasmic IKK complex. Thus, NEMO represents a common nuclear signal that mediates the nuclear-to-cytoplasmic signaling pathway induced by these distinct anticancer agents. Through the following Specific Aims, we will test the hypothesis that a series of NEMO posttranslational modifications mediates its nuclear import and export to chaperone an IKK regulator from the nucleus to the cytoplasm to promote IKK activation in response to genotoxic stimuli. Aim 1: Specify the mechanism of SUMO-1 modification of NEMO by genotoxic agents. Aim 2: Determine the role of ubiquitin in nuclear export of NEMO in genotoxic signaling. Aim 3: Elucidate the role of ATM in IKK activation by genotoxic agents. The proposed studies will help determine the mechanisms fundamental to NF-KB activation by certain genotoxic anticancer agents. These mechanisms may also serve as models for activation pathways induced by other genotoxic agents, such as ionizing radiation, and other agents that target DNA topoisomerase I or II. Moreover, new reagent tools, such as phospho-specific NEMO antibodies, cDNAs encoding wild type and mutant versions of SUMO and ubiquitin ligases for NEMO, and cell systems that harbor these mutant proteins, will also be useful for other investigators in their efforts to dissect NF-KB activation pathways. Finally, specific enzymes and processes involved in this activation pathway may serve as rational therapeutic targets to induce chemo/radio-sensitization in certain types of human cancer.